Clostridial neurotoxins are neurotoxins secreted from the Clostridium genus of bacteria. Such neurotoxins are potent inhibitors of calcium-dependent neurotransmitter secretion in neuronal cells. They are considered to mediate this activity through a specific endoproteolytic cleavage of at least one of the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) proteins, which include VAMP (synaptobrevin, cellubrevin), syntaxin, and SNAP-25. SNARE proteins are thought to be central to the vesicle docking and membrane fusion events of neurotransmitter secretion or hormone secretion. The neuronal cell targeting of clostridial neurotoxins, such as tetanus neurotoxins and botulinum neurotoxins, is considered to be a receptor-mediated event following which the toxins become internalized and subsequently traffic to the appropriate intracellular compartment where they affect their protease activity.
Botulinum neurotoxin and tetanus neurotoxin are expressed as 150-kDa single polypeptides (termed dichains) containing a disulfide bond between the 50-kDa N-terminal light chain (LC) and the 100-kDa C-terminal heavy chain (HC) (FIG. 1). A post-translational cryptic cleavage generates the mature toxin structure consisting of two chains connected by a disulfide bond. The LC contains the zinc-protease catalytic domain, responsible for the toxins' intracellular enzymatic activity. The 100-kDa HC may be further proteolyzed into a 50-kDa N-terminal membrane-spanning domain (Hn) and a 50-kDa C-terminal receptor-binding domain (Hc).
The molecular mechanism of toxin intoxication of these neurotoxins appears to involve at least three steps or stages. In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the HC and a cell surface receptor. The receptor is thought to be different for each serotype of botulinum toxin and for tetanus toxin. The carboxyl end segment of the HC, Hc, appears to be important for targeting of the toxin to the cell surface.
In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This last step is thought to be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra-endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin then translocates through the endosomal membrane into the cytosol.
The last step of the mechanism of neurotoxin activity appears to involve reduction of the disulfide bond joining the HC and LC. The entire toxic activity of botulinum and tetanus toxins is contained in the LC of the holotoxin. The LC is a zinc (Zn2+) protease, which then selectively cleaves specific sites of one of the three SNARE proteins. Their proteolysis inhibits exocytosis and blocks acetylcholine secretion, leading ultimately to muscular paralysis. The LC itself is non-toxic because it cannot translocate through cholinergic nerve endings into cytosol.
Botulinum neurotoxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin type A has been approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus, and hemifacial spasm. Non-type A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to botulinum toxin type A. Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A averages about three months.